BAX and BAK are pro-apoptotic BCL-2 family proteins that function as the essential gatekeepers of mitochondrial apoptosis. In response to cellular stress, BAX and BAK transform from inactive monomers into toxic, homo-oligomeric pores that pierce the mitochondrial membrane, releasing apoptogenic factors that drive the death pathway. Anti-apoptotic proteins, such as BCL-2, block cell death by trapping the critical death effector helix, termed BH3, of BAX and BAK in a surface groove. The BH3-only subgroup of BCL-2 proteins only share a conserved BH3 domain, which is deployed to transmit stress signals to anti- and pro-apoptotic targets. Whereas BH3-only proteins can bind the anti-apoptotic groove to derepress activated conformers of BAX and BAK (indirect activation), select members can also directly trigger BAX and BAK (direct activation). The structural and biochemical basis for the direct activation mechanism was long unknown. In the current award period, we demonstrated that chemically-reinforced BH3 helices of the activator BH3-only proteins BIM, BID, and PUMA bound to a novel trigger site on the N-terminal face of BAX, directly activating BAX-mediated mitochondrial membrane poration and cellular apoptosis. This work revealed a new interaction paradigm for BCL-2 family proteins, and has formed the basis for designing prototype therapeutics to reactivate apoptosis in cancer through direct BAX engagement. Facilitated by our development of a recombinant form of full-length BAK and a photoaffinity labeling/mass spectrometry method for rapid binding-site identification, we recently identified functional BH3 interaction sites at the C-terminal faces of BAK and BAX. We hypothesize that these C-terminal sites propel BAX and BAK activation at the mitochondrial membrane. Thus, we now propose to develop specific probes for the three BH3 trigger sites to systematically dissect the structural, biochemical, and functional consequences of direct and selective BAX/BAK activation. Specifically, we aim to (1) dissect the binding and specificity determinants for direct engagement of the BH3 trigger sites of pro-apoptotic BAX and BAK, (2) functionally link the selective BAX and BAK binding activity of BH3 helices with direct activation of BAX/BAK-mediated membrane poration in vitro and in cells, and (3) define the conformational changes along the direct BAX and BAK activation pathways by hydrogen-deuterium exchange mass spectrometry (HXMS). By complementing our biochemical and functional studies with structural determination of the three BH3/trigger site complexes, we aim to provide new blueprints for the development of pharmacologic activators of apoptosis through direct BAX and BAK triggering. We will also apply HXMS to accomplish the first BAX/BAK-in-motion study of its kind, designed to unveil the continuum of structural changes from monomer to oligomer in real time and in the physiologic membrane environment. In combining chemistry, biochemistry, structural biology, mass spectrometry, and apoptosis biology, we remain laser-focused on elucidating the fundamental protein interaction mechanisms harnessed by BAX and BAK to execute mitochondrial apoptosis.